Method for analyzing protein-protein interaction on single-molecule level in cell environment, and method for measuring density of protein activated in cytosol

ABSTRACT

A method of analyzing protein-protein interactions at a single molecular level is disclosed. The method of analyzing the interactions between first proteins and second proteins at the single molecular level includes: preparing at least two substrates, in which first protein-binding molecules that are biomolecules to be bound to the first proteins are attached to each of the substrates; inducing binding between the first proteins and the first protein-binding molecules on the first substrate and the second substrate, respectively, by supplying the first proteins included in the control group-cell to the first substrate among the two substrates and supplying the first proteins included in the experimental group-cell to the second substrate among the two substrates; supplying cell lysates of cells including the marker-tagged second proteins to the first substrate and the second substrate, respectively, when the first proteins and the first protein-binding molecules are bound to the first substrate and the second substrate, respectively; and comparing and analyzing the interactions between the first proteins and the second proteins on the first substrate and the second substrate in the supply of the cell lysates to the first substrate and the second substrate, respectively. For observing the interactions between the first proteins and the second proteins, the state of each cell and activation levels of the first proteins can be compared and analyzed by comparing after varying of a type of cells supplying the first proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/059,294 filed on October 21 (now U.S. Pat. No. 9,377,462)and claims the benefit of U.S. patent application Ser. No. 14/059,294(now U.S. Pat. No. 9,377,462), which are incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to a method of analyzing protein-proteininteractions and a method of measuring activated protein concentrationin a cell lysate, and more specifically, to a method of analyzingprotein-protein interactions, which can analyze the protein-proteininteractions at a single molecular level in the actual intracellularenvironment, and also can compare and confirm each cell state andactivation levels of first proteins by comparing after varying of a typeof cell supplying the first proteins for observing interactions betweenthe first proteins and the second proteins, and a method of measuringactivated protein concentration in a cell lysate, which canquantitatively compare and measure specific activated proteinconcentrations in the cell of an experimental group and the cell of acontrol group.

BACKGROUND ART

A cell maintains a life phenomenon by performing several biologicalfunctions, such as gene expression, cell growth, cell cycle, metabolism,signal transduction, and the like, through various and complexprotein-protein interactions. Accordingly, the understandings ofintracellular protein-protein interactions and functions of theinteractions have been the foundation of the understandings of the lifephenomenon and are an essential part for developing new drugs andtreating diseases.

A representative method of investigating protein-protein interactions invitro is an affinity chromatography method.

In the case of protein affinity chromatography, it is difficult topurify a protein. In addition, since the interactions between proteinsare confirmed only in vitro, it may result in false-positive results forproteins to be bound by an electrostatic interaction during passing theproteins, which are not interacted in the cell, through a column.

That is, in order to perform a quantitative measurement, a method ofinvestigating the protein-protein interactions according to theconventional technologies analyzes the interactions in the isolation ofproteins from other intracellular materials by purifying each of theproteins that exist in the cell for analyzing the protein-proteininteractions. Accordingly, there is a limit to analyze theprotein-protein interactions at the single molecular level in the actualintracellular environment with co-existing other proteins, and the like.

Moreover, a method of investigating the protein-protein interactionsaccording to the conventional technologies has a problem that thedegrees of effects of other proteins on specific protein-proteininteractions cannot be analyzed when other proteins are involved withthe interactions in the actual intracellular environment.

In addition, there was a conventional limit to compare and measurequantitatively specific activated protein concentrations in the cell ofan experimental group and the cell of a control group.

SUMMARY OF THE DISCLOSURE

Accordingly, an object of the present invention is to provide a methodof analyzing protein-protein interactions, which can analyze theprotein-protein interactions at a single molecular level in the actualintracellular environment, as well as can compare and confirm each cellstate and activation levels of first proteins by comparing after varyingof a type of cells supplying the first protein for observinginteractions between the first proteins and the second proteins.

Moreover, another object of the present invention is to provide a methodof measuring activated protein concentrations in a cell lysate, whichcan compare and measure quantitatively specific activated proteinconcentrations in the cell of an experimental group and the cell of acontrol group.

To accomplish the above objects, according to one aspect of the presentinvention, there is provided a method of analyzing interactions betweenfirst proteins and second proteins by at a single molecular level,comprising: (a) preparing at least two substrates, in which firstprotein-binding molecules that are biomolecules to be bound to the firstproteins are attached to each of the substrates; (b) inducing bindingbetween the first proteins and the first protein-binding molecules onthe first substrate and the second substrate, respectively, by supplyingthe first proteins included in the control group-cell to the firstsubstrate among the two substrates and supplying the first proteinsincluded in the experimental group-cell to the second substrate amongthe two substrates; (c) supplying cell lysates of cells including themarker-tagged second proteins to the first substrate and the secondsubstrate, respectively, when the first proteins and the firstprotein-binding molecules are bound to the first substrate and thesecond substrate, respectively; and (d) comparing and analyzing theinteractions between the first proteins and the second proteins on thefirst substrate and the second substrate in the supply of the celllysates to the first substrate and the second substrate, respectively.

Preferably, in the step (b), the control group-cell is a normal cell andthe experimental group-cell is a tumor cell.

Preferably, the step (d) includes measuring a fluorescent signal havinga specific wavelength generated by the markers tagged to the secondproteins bound to the first proteins using an optical apparatusgenerating a near-field.

Preferably, in the step (d), the fluorescent signal having the specificwavelength is cumulatively measured for a predetermined time period.

Preferably, in the step (d), the fluorescent signal having the specificwavelength is measured in real-time under the presence of the celllysates supplied to the substrate.

Preferably, the step (b) includes supplying the cell lysates of thecontrol group-cell to the first substrate and supplying the cell lysatesof the experimental group-cell to the second substrate.

Preferably, the method further comprises supplying a buffer solution tothe substrate before the step (c).

According to another aspect of the present invention, there is provideda method of measuring activated protein concentration in a cell lysate,comprising: (a) preparing a substrate attaching first protein-bindingmolecules that are biomolecules to be bound with first proteins; (b)inducing binding of the first proteins and the first protein-bindingmolecules by supplying the cell lysates including the first proteins tothe substrate; (c) supplying the cell lysates including themarker-tagged second proteins when the first proteins are bound to thefirst protein-binding molecules on the substrate; and (d) analyzinginteractions between the first proteins and the second proteins on thesubstrate.

Preferably, the method further comprises (e) repeating the steps (b) to(d) by increasing the concentration of the cell lysates including thefirst proteins in the step (b), by a predetermined ratio.

Preferably, the step (d) includes measuring a generation frequency of afluorescent signal having a specific wavelength generated by the markerstagged to the second proteins bound to the first proteins in anyconfigured observation region on the substrate.

Preferably, the first proteins that interact with the second proteins inthe step (d) are activated first proteins among the first proteins boundto the first protein-binding molecules.

Preferably, the step (d) includes measuring a fluorescent signal havinga specific wavelength generated by the markers tagged to the secondproteins bound to the first proteins using an optical apparatusgenerating a near-field.

According to yet another aspect of the present invention, there isprovided a method of measuring activated protein concentration in a celllysate, comprising: (a) preparing at least two substrates, to whichfirst protein-binding molecules that are biomolecules to be bound tofirst proteins are attached, respectively; (b) inducing binding betweenthe first proteins and the first protein-binding molecules on the firstsubstrate and the second substrate, respectively, by supplying the firstproteins included in the control group-cell to the first substrate amongthe two substrates and supplying the first proteins included in theexperimental group-cell to the second substrate among the twosubstrates; (c) supplying cell lysates including the marker-taggedsecond proteins to the first substrate and the second substrate,respectively, when the first proteins and the first protein-bindingmolecules are bound to the first substrate and the second substrate,respectively; and (d) comparing and analyzing the interactions betweenthe first proteins and the second proteins on the first substrate andthe second substrate in the supply of the cell lysates to the firstsubstrate and the second substrate, respectively.

Preferably, the method further comprises (e) repeating the steps (b) to(d) while increasing the concentrations of the control group-cell lysateand the experimental group-cell lysate in the step (b), by apredetermined ratio.

Preferably, the step (d) includes comparing and measuring a generationfrequency of a fluorescent signal having a specific wavelength generatedby the markers tagged to the second proteins bound to the first proteinsin any configured observation region on the substrate.

Preferably, the first proteins that interact with the second proteins inthe step (d) are activated first proteins among the first proteins boundto the first protein-binding molecules.

Preferably, the step (d) includes measuring a fluorescent signal havinga specific wavelength generated by the markers tagged to the secondproteins bound to the first proteins using an optical apparatusgenerating a near-field.

According to the present invention, it is possible to analyze theprotein-protein interactions at a single molecular level in the actualintracellular environment, as well as to compare and confirm each cellstate and activation levels of first proteins by comparing after varyingof a type of cells supplying the first protein for observinginteractions between the first proteins and the second proteins.

Moreover, according to the present invention, it possible to provide amethod of measuring activated protein concentrations in a cell lysate,which can compare and measure quantitatively specific activated proteinconcentrations in the cell of an experimental group and the cell of acontrol group.

BRIEF DESCRIPTION OF DRAWINGS

The above and other objects, features and advantages of the presentinvention will become more apparent to those of ordinary skill in theart by describing in detail exemplary embodiments thereof with referenceto the attached drawings, in which:

FIG. 1 is a flowchart illustrating a procedure of a method of analyzingprotein-protein interactions according to an embodiment of the presentinvention;

FIG. 2 to FIG. 3 are diagrams illustrating each step of a method ofanalyzing the protein-protein interactions according to an embodiment ofthe present invention;

FIG. 4 is a diagram illustrating a changing process of a cell state inanother embodiment of the present invention;

FIG. 5 is a flowchart of the procedure illustrating a method ofanalyzing protein-protein interactions according to another embodimentof the present invention;

FIG. 6 is a graph showing signals observed at a first substrate bindingfirst proteins in the cell of a control group;

FIG. 7 is a graph showing signals observed at a second substrate bindingfirst proteins in the cell of an experimental group;

FIG. 8 is a diagram illustrating a method of comparing activated proteinconcentrations in the cell of a control group and the cell of anexperimental group according to another embodiment of the presentinvention;

FIG. 9 is a diagram showing an experimental result of measuringinteractions between first proteins and second proteins while graduallyincreasing the concentrations of the first proteins included in the cellof an experimental group;

FIG. 10 and FIG. 11 are graphs showing measurements of fluorescentsignals measured with increasing concentrations of the first proteinsincluded in the cell of an experimental group;

FIG. 12 and FIG. 13 are graphs showing measurements of fluorescentsignals measured with increasing concentrations of the first proteinsincluded in the cell of a control group; and

FIG. 14 is a graph showing comparisons of changes of frequenciesmeasured with increasing concentrations of the first proteins in thecell of an experimental group and the cell of a control group,respectively.

DETAILED DESCRIPTION

Hereinafter, exemplary embodiments of the present invention will bedescribed in detail. However, the present invention is not limited tothe embodiments disclosed below, but can be implemented in variousforms. The following embodiments are described in order to enable thoseof ordinary skill in the art to embody and practice the presentinvention.

With reference to the appended drawings, exemplary embodiments of thepresent invention will be described in detail below. To aid inunderstanding the present invention, like numbers refer to like elementsthroughout the description of the figures, and the description of thesame elements will be not reiterated.

FIG. 1 is a flowchart illustrating a procedure of a method of analyzingprotein-protein interactions according to an embodiment of the presentinvention. Referring to FIG. 1, for a method of analyzing theprotein-protein interactions according to an embodiment of the presentinvention, firstly, an analyst attaches first protein-binding antibodies(Primary antibody) that are antibodies to be bound to the first proteinsto a substrate that is a quartz slide coated with polyethylene glycol(PEG), in order to analyze the interactions between the first proteinsand the second proteins that are two specific proteins at a singlemolecular level (S110).

For the experiment according to the present invention, the firstproteins are h-Ras proteins and the second proteins are Ras-bindingdomain (RBD) proteins of C-Raf.

Meanwhile, according to an embodiment of the present invention, theantibodies to be bound to the first proteins may include biomolecules tobe bound to the first proteins, such as DNA, RNA, or liposomes havingspecific components to be bound to proteins, and the like, in additionto antibodies.

Subsequently, the analyst induces bindings between the first proteinsand the first protein-binding antibodies by supplying (S120) cytoplasmiclysate of the cell including the first proteins to the substrate (S130).

Meanwhile, for performing the above-described step, S120, cell lysates,such as cytolysate, diluted cytoplasmic lysate, diluted or purifiedcytolysate, and the like, may be used in addition to the cytoplasmiclysate.

Meanwhile, according to an embodiment of the present invention, theanalyst may pre-treat for an expression of predetermined firstfluorescent proteins, such as m-Cherry, and the like, to the firstproteins. In this case, when the first protein-binding antibodies arebound to the first proteins, the analyst may confirm whether the firstproteins are bound to a plurality of first protein-binding antibodiesattached on the substrate from the change of wavelength by the firstfluorescent proteins that are expressed to the first proteins (i.e.,measuring individual single molecule signal generated from the firstfluorescent proteins) by performing an observation of the surface of thesubstrate using a total internal reflection fluorescence microscope.

That is, when the first fluorescent proteins are expressed in the firstproteins, whether or not the first proteins are bound to the antibodiesmay be confirmed through the total internal reflection fluorescencemicroscope, and thus the number of the first proteins bound to theantibodies attached on the substrate and binding density thereof may beaccurately measured.

Once confirming that the first proteins are bound to a plurality offirst protein-binding antibodies attached on the substrate,respectively, the analyst removes the remaining materials included inthe cytoplasmic lysate except the first proteins from the substrate bysupplying a buffer solution to the substrate (S140).

Subsequently, the analyst manipulates for an expression of secondfluorescent proteins through a genetic manipulation of second proteinsbeing in different relevant cells among the same cells as theabove-described cells (S150). Meanwhile, according to an embodiment ofthe present invention, the second fluorescent molecules may be attachedor connected to the second proteins by a physical-chemical method.

Meanwhile, the above described step, S150, may be performed in advancebefore the above-described step, S110 in order to smoothly progress theanalysis. According to an embodiment of the present invention, since thesecond fluorescent proteins preferably have wavelength range differentfrom the first fluorescent proteins, when the first fluorescent proteinsare m-Cherry proteins, the second fluorescent proteins may be eGFPs(enhanced Green Fluorescent Protein) that is a green fluorescentprotein.

Subsequently, the analyst supplies the whole cytoplasmic lysate of thecell having the expressed second fluorescent proteins inside the secondproteins from the above-described step, S150, to the substrate (S160).

At least part of the plurality of the first protein-binding antibodiesattached on the surface of the substrate is bound to each first protein.When the whole cytoplasmic lysate including the second proteins issupplied to the above-described surface of the substrate as shown inFIG. 3, the first proteins (Cellular Ras) on the surface of thesubstrate interact with the second proteins in the same condition as theintracellular environment in the coexisting with the second proteins(eGFP-cRBD) included in the cytoplasmic lysate and native proteins inthe whole cell lysate.

In short, as shown in FIG. 3, each first protein (Cellular Ras) bound toeach antibody (Anti-Ras Primary antibody) attached on the surface of thesubstrate interacts with the second protein (eGFP-cRBD) in the samecondition as the intracellular environment at the single molecular levelthereby repeating binding and the unbinding.

Meanwhile, for performing the above-described step, S160, cell lysates,such as cytolysate, diluted cytoplasmic lysate, diluted or purifiedcytolysate, and the like, may be used in addition to the cytoplasmiclysate.

As shown in FIG. 3, when the first proteins are bound to the secondproteins at the single molecular level, the analyst can confirm that thewavelength on the surface of the substrate is changed from 473 nm to 520nm by eGFP that is a fluorescent protein that is expressed to the secondproteins through performing an observation of substrate surface usingthe total internal reflection fluorescence microscope having 473 nmwavelength.

That is, the binding state between the first proteins and the secondproteins can be confirmed through a detection of fluorescence signalshaving a specific wavelength bandwidth (520 nm) generated from thesecond fluorescent protein (eGFP) located on the surface of thesubstrate through the binding between the first proteins and the secondproteins, and the analyst can analyze the interactions, such as thefrequencies of binding and unbinding between the first proteins and thesecond proteins, and the like, at the single molecular level bycontinuously observing the change of wavelength at each of antibodiesattached on the surface of the substrate (S170).

In addition, the interactions, such as the frequencies of the bindingand unbinding between the first proteins and the second proteins, andthe like, can be analyzed in the same environment as the intracellularenvironment by measuring the fluorescence signals having a specificwavelength in real time under the presence of the cytoplasmic lysatesupplied to the substrate in the above-described step, S160.

Meanwhile, according to an embodiment of the present invention, theanalyst may compare and analyze the interactions between the firstproteins and the second proteins in the cell of a normal state (cell ofa control group) and the interactions between the first proteins and thesecond proteins in cell of an abnormal state (cell of an experimentalgroup).

According to an embodiment of the present invention, a normal breasttissue cell is changed to a tumor cell as shown in FIG. 4 byartificially maximizing an activation of Ras protein in the cellisolated from a breast tissue of a human body, and the normal breasttissue cell was used as the cell of a control group and the breasttissue cell to be changed to the tumor cell was used as the cell of anexperimental group.

FIG. 5 is a flowchart illustrating a procedure of a method of analyzingprotein-protein interactions according another embodiment of the presentinvention. Referring to FIG. 5, for a method of analyzing theprotein-protein interactions according to another embodiment of thepresent invention, firstly, the analyst prepares two quartz slidesubstrates with the same size coated with polyethylene glycol (PEG).

Subsequently, the analyst attaches the first protein-binding antibodies(Anti-Ras Primary antibody) that are the antibodies to be bound to thefirst proteins on both of two substrates (first substrate and secondsubstrate) as shown in FIG. 2 (S210).

Meanwhile, according to an embodiment of the present invention, theantibody to be bound to the first proteins may include biomolecules tobe bound to the first proteins, such as DNA, RNA, or liposomes havingspecific components to be bound to proteins, and the like, in additionto antibodies.

Subsequently, the analyst induces binding between the first proteins andthe first protein-binding antibodies (Anti-Ras Primary antibody) on eachof the first substrate and the second substrate by supplying (S220) thecytoplasmic lysate of the cell of the control group including the firstproteins to the first substrate and the cytoplasmic lysate of the cellof the experimental group including the first proteins to the secondsubstrate (S230).

Meanwhile, according to an embodiment of the present invention, theconcentration of the first proteins included in the cell of the controlgroup should be equal to the concentration of the first proteinsincluded in the cell of the experimental group. To achieve this, itshould be confirmed whether both of the concentrations of the firstproteins included in the cells of the control group and the experimentalgroup are one and the same by measuring both of the concentrations. Whenboth of the concentrations are not the same as each other, theabove-described step, S230, should be performed after both of theconcentrations are adjusted to be the same as each other throughdiluting, and the like.

At this time, various methods of measuring protein concentrationaccording to the conventional technologies may be used for measuring theprotein concentration, and a method of measuring protein concentrationas disclosed in Korean Patent Application No. 10-2011-0088074(Applicant, Tae-Young Yoon) may be also used. Meanwhile, the presentdescription includes the description and figures as shown in KoreanPatent Application No. 10-2011-0088074 as a part thereof.

Meanwhile, for performing the above-described step, S230, cell lysates,such as cytolysate, diluted cytoplasmic lysate, diluted or purifiedcytolysate, and the like, may be used in addition to the cytoplasmiclysate.

Meanwhile, according to an embodiment of the present invention, theanalyst may pre-treat for an expression of predetermined firstfluorescent proteins, such as m-Cherry, and the like, to the firstproteins. In this case, when the first protein-binding antibodies arebound to the first proteins, the analyst may confirm whether the firstproteins are bound to the plurality of the first protein-bindingantibodies attached on the substrate from the change of wavelength bythe first fluorescent proteins that are expressed in the first proteins(i.e., measuring single molecule signal generated from the firstfluorescent proteins) by performing an observation of the surface of thesubstrate using the total internal reflection fluorescence microscope.

That is, when the first fluorescent proteins are expressed in the firstproteins, whether or not the first proteins are bound to the antibodiesmay be confirmed through the total internal reflection fluorescencemicroscope, and thus the number of the first proteins bound to theantibodies attached on the substrate and binding density thereof may beaccurately measured.

When it is confirmed that the first proteins are bound to the pluralityof the first protein-binding antibodies attached on the first substrateand the second substrate, respectively, the analyst removes theremaining materials included in the cytoplasmic lysate except the firstproteins from the substrates by supplying a buffer solution to thesubstrates (S240).

Subsequently, the analyst manipulates to express second fluorescentproteins through a genetic manipulation of second proteins that exist inspecific cells (S250). Meanwhile, according to an embodiment of thepresent invention, the second fluorescent proteins may be attached orconnected to the second proteins by a physical-chemical method.

Meanwhile, the above-described step, S250, may be performed in advancebefore the above-described step, S210 in order to smoothly progress theanalysis. According to an embodiment of the present invention, thesecond fluorescent proteins may preferably have a wavelength regiondifferent from that of the first fluorescent proteins, and thus when thefirst fluorescent proteins are m-Cherry proteins, the second fluorescentproteins may be eGFP (enhanced Green Fluorescent Protein) that is agreen fluorescent protein.

Subsequently, the analyst supplies the whole cytoplasmic lysates of thecell with the expressed second fluorescent proteins inside the secondproteins in the above-described step, S260, to the first substrate andthe second substrate, respectively (S260).

Meanwhile, for performing the above-described step, S260, theconcentration of the second proteins in the cytoplasmic lysates to besupplied to the first substrate and the second substrate, respectively,should be the same as each other. To achieve this, it should beconfirmed whether both of the concentrations of the second proteinsincluded in two cytoplasmic lysates are one and the same by measuringboth of the concentrations. When both of the concentrations are not thesame as each other, the above-described step, S260, should be performedafter both of the concentrations are adjusted to be the same as eachother through diluting, and the like.

At this time, various methods of measuring protein concentrationaccording to the conventional technologies may be used for measuring theprotein concentration, and a method of measuring protein concentrationas above-disclosed in Korean Patent Application No. 10-2011-0088074(Applicant, Tae-Young Yoon) may be also used.

Meanwhile, for performing the above-described step, S220, cell lysates,such as cytolysate, diluted cytoplasmic lysate, diluted or purifiedcytolysate, and the like, may be used in addition to the cytoplasmiclysate.

The first proteins of the cell of the control group and the firstproteins of the cell of the experimental group are bound to theplurality of the first protein-binding antibodies that are attached tothe surfaces of the first substrate and the second substrate,respectively. When the whole cytoplasmic lysate including the secondproteins on the above-mentioned surface of the substrate are supplied asshown in FIG. 3, the first proteins on each surface of the substratesinteract with the second proteins in the same state as the intracellularenvironment that co-exists with the second proteins (eGFP-cRBD) includedin the cytoplasmic lysate and native proteins in the whole cell lysate.

The analyst may confirm binding state of the first proteins and thesecond proteins through a detection of the fluorescent signal having aspecific wavelength bandwidth (520 nm) generated from the secondfluorescent protein (eGFP) to be located on the surface of the substratethrough binding between the first proteins and the second proteins, andmay compare and analyze the interactions, such as the frequencies ofbinding and unbinding between the first proteins and the second proteinson the first substrate and the second substrate, and the like, bycontinuously observing the change of wavelength of each antibodyattached on the surface of the substrate (S270).

FIG. 6 is a graph showing the signal observed on the first substratebound with the first proteins in the cell of the control group and FIG.7 is a graph showing the signal observed on the second substrate boundwith the first proteins in the cell of the experimental group.

Comparing between FIG. 6 and FIG. 7, it may be confirmed that theinteraction between the first proteins and the second proteins in thecell of the experimental group, that is a tumor cell, is more active ascompared with the interaction between the first proteins and the secondproteins in the cell of the control group, that is a normal cell. Fromthis, the analyst may confirm that Ras protein that is the first proteinin the tumor cell is excessively activated

Meanwhile, the first proteins interacting with the second proteins maybecome to be the activated first proteins, and thus the activationlevels of the first proteins in the cell of a control group and thefirst proteins in the cell of a experimental group may be quantitativelymeasured by comparing the interactions between the first proteins andthe second proteins in the cell of the control group and theinteractions between the first proteins and the second proteins in thecell of the experimental group as mentioned above.

To achieve this, the present invention uses the method as shown in FIG.8. FIG. 8 is a diagram illustrating a method of comparing the activatedprotein concentrations in the cells of a control group and anexperimental group according to another embodiment of the presentinvention.

Referring to FIG. 8, the analyst prepares two quartz slide substrateswith the same size coated with polyethylene glycol (PEG) and attachesthe first protein-binding antibodies (Anti-Ras Primary antibody) thatare antibodies to be bound to each first protein to two substrates(first substrate and second substrate) as shown in FIG. 2 (S310).

Subsequently, the analyst induces binding between the first proteins andthe first protein-binding antibodies (Anti-Ras Primary antibody) on eachof the first substrate and the second substrate, respectively, bysupplying (S320) the cell lysates of the control group-cell includingthe first proteins to the first substrate and supplying (S330) the celllysates of the experimental group-cell including the first proteins tothe second substrate.

Meanwhile, according to an embodiment of the present invention, theconcentration of the first proteins included in the cell of the controlgroup should be equal to the concentration of the first proteinsincluded in the cell of the experimental group. To achieve this, itshould be confirmed whether both of the concentrations of the firstproteins included in the cells of the control group and the experimentalgroup are one and the same by measuring both of the concentrations.

When both of the concentrations are not the same as each other, theabove-described steps, S320 to S220, should be performed after both ofthe concentrations are adjusted to be the same as each other throughdiluting, and the like.

At this time, various methods of measuring protein concentrationaccording to the conventional technologies may be used for measuring theprotein concentration, and a method of measuring protein concentrationas above-disclosed in Korean Patent Application No. 10-2011-0088074(Applicant, Tae-Young Yoon) may be also used.

Meanwhile, according to an embodiment of the present invention, theanalyst may pre-treat for an expression of predetermined firstfluorescent proteins, such as m-Cherry, and the like, to the firstproteins. In this case, when the first protein-binding antibodies arebound to the first proteins, the analyst may confirm whether the firstproteins are bound to the plurality of the first protein-bindingantibodies attached on the substrate from the change of wavelength bythe first fluorescent proteins that are expressed to the first proteins(i.e., measuring single molecule signal generated from the firstfluorescent proteins) by performing an observation of the surface of thesubstrate using the total internal reflection fluorescence microscope.

That is, when the first fluorescent proteins are expressed in the firstproteins, whether the first proteins are bound to the antibodies may beconfirmed through the total internal reflection fluorescence microscope,and thus the number of the first proteins bound to the antibodiesattached on the substrate and binding density thereof may be accuratelymeasured.

When it is confirmed that the first proteins are bound to the pluralityof the first protein-binding antibodies attached on the first substrateand the second substrate, respectively, the analyst removes theremaining materials included in the cytoplasmic lysate except the firstproteins from the substrates by supplying a buffer solution to thesubstrates.

Subsequently, the analyst manipulates to express second fluorescentproteins through a genetic manipulation of second proteins that existfor specific cells. Meanwhile, according to an embodiment of the presentinvention, the second fluorescent proteins may be attached or connectedto the second proteins by a physical-chemical method.

Meanwhile, according to an embodiment of the present invention, thesecond fluorescent proteins may preferably have a wavelength regiondifferent from that of the first fluorescent proteins, and thus when thefirst fluorescent proteins are m-Cherry proteins, the second fluorescentproteins may be eGFP (enhanced Green Fluorescent Protein) that is agreen fluorescent protein.

Subsequently, the analyst supplies the whole cell lysate of the cellwith the expressed second fluorescent proteins inside the secondproteins to the first substrate and the second substrate, respectively(S340).

Meanwhile, for performing the above-described step, S340, theconcentration of the second proteins in the cell lysate to be suppliedto the first substrate and the second substrate, respectively, should bethe same as each other. To achieve this, it should be confirmed whetherboth of the concentrations of the second proteins included in twocytoplasmic lysates are one and the same by measuring both of theconcentrations. When both of the concentrations are not the same as eachother, the above-described step, S340, should be performed after both ofthe concentrations are adjusted to be the same as each other throughdiluting, and the like.

At this time, various methods of measuring protein concentrationaccording to the conventional technologies may be used for measuring theprotein concentration, and a method of measuring protein concentrationas above-disclosed in Korean Patent Application No. 10-2011-0088074(Applicant, Tae-Young Yoon) may be also used.

The first proteins of the cell of the control group and the firstproteins of the cell of the experimental group are bound to theplurality of the first protein-binding antibodies that are attached tothe surfaces of the first substrate and the second substrate,respectively. When the whole cell lysates, such as the cytoplasmiclysate including the second proteins on the above-mentioned surface ofthe substrate, and the like, are supplied as shown in FIG. 3, the firstproteins on each surface of the substrates interact with the secondproteins in the same state as the intracellular environment that allowsfor the coexistence of the second proteins (eGFP-cRBD) included in thecytoplasmic lysate and native proteins in the whole cell lysate.

The analyst may confirm binding state of the first proteins and thesecond proteins through a detection of the fluorescent signal having aspecific wavelength bandwidth (520 nm) generated from the secondfluorescent protein (eGFP) to be located on the surface of the substratethrough binding between the first proteins and the second proteins, andmay compare and analyze the interactions, such as the frequencies ofbinding and unbinding between the first proteins and the second proteinson the first substrate and the second substrate, and the like, bycontinuously observing the change of wavelength to each antibodyattached on the surface of the substrate (S350).

More specifically, the analyst repeats the above-described steps, S320to S350, while gradually and equally increasing the concentration of thefirst proteins included in the cell lysate of the control group and theconcentration of the first proteins included in the cell of theexperimental group supplied to the first substrate and the secondsubstrate in the above-described steps, S320 and S330.

FIG. 9 is a diagram showing the experimental results of measuring theinteraction between the first proteins and the second proteins withgradually increasing the concentration of the first proteins included inthe cell of the experimental group. Referring to FIG. 9(a), the celllysate of the cell of the experimental group including the firstproteins (HRas) of 1 nM concentration is supplied to the secondsubstrate to induce binding between the first proteins and the firstprotein-binding molecules and then the generated signal of thefluorescent signal having a specific wavelength generated by markerstagged to the second proteins is measured by using an optical apparatusgenerating a near-field in the supply of the cell lysates including themarker-tagged second proteins to the second substrate.

Specifically, since only activated first proteins among the firstproteins bound to the first protein-binding molecules attached to thesecond substrate interact with the second protein, for measuring usingthe above-described optical apparatus, the optical apparatus can measurea frequency of fluorescent signal generation with each ROI (Region ofInterest) as the center after setting ROI of any 1.1 μm² size with thepoint of sensing the fluorescent signal having a specific wavelengththat exceeds a predetermined threshold as the center.

Meanwhile, referring to FIG. 9(b), the cell lysate of the cell of theexperimental group including the first proteins (HRas) of 2 nMconcentration is supplied to the second substrate to induce bindingbetween the first proteins and the first protein-binding molecules andthen the generated signal of the fluorescent signal with a specificwavelength generated by a marker tagged to the second proteins ismeasured by using an optical apparatus generating a near-field in thesupply of the cell lysate including the marker-tagged second proteins tothe second substrate.

Specifically, since only activated first proteins among the firstproteins bound to the first protein-binding molecules attached to thesecond substrate interact with the second protein, when theconcentration of the cell lysate of the cell of the experimental groupincluding the first proteins is increased by two times, the point ofsensing the fluorescent signal of a specific wavelength that exceeds apredetermined threshold is observed by two times as compared with FIG.9(a) (Four ROI→Eight ROI).

Meanwhile, since the concentration of the cell lysate of theexperimental group-cell is not still relatively high, only one of pairsof the interactions between the activated first proteins and secondproteins in one ROI having a certain size is being observed.

Accordingly, the frequency of the fluorescent signal generationaveragely measured in eight ROI in FIG. 9(b) is equal to that of FIG.9(a).

However, since a configurable number of ROI according to the size of thesecond substrate is limited, when the concentration of the experimentalgroup-cell lysate including the first proteins exceeds at least athreshold, at least two pairs of the interactions between activatedfirst proteins and second proteins are observed in one ROI as shown inFIG. 9(c).

When the concentration of the experimental group-cell lysate exceeds athreshold as mentioned above, the average value of the frequency of thefluorescent signal generation becomes to increase in each ROI and thenthe average value of the frequency of the fluorescent signal generationis continuously increased with an increasing concentration of theexperimental group-cell lysate, continuously.

According to an embodiment of the present invention, when the sizes ofthe first substrate and second substrate are 45×90 μm², the threshold ofthe experimental group-cell lysate of the tumor cell including the firstproteins (HRas), in which the average value of the frequency of thefluorescent signal generation is started to increase in each ROI withthe size of 1.1 μm², is measured to be 5 nM.

That is, as confirmed in FIG. 10, the cell lysate of the experimentalgroup-cell including the first proteins (HRas) with 3 nM concentrationis supplied to the second substrate to induce binding between the firstproteins and the first protein-binding molecules. From that point, whenthe frequency of the fluorescent signal generation with specificwavelength generated by the markers tagged to the second proteins ismeasured by using an optical apparatus in a plurality of ROIs as itsaverage value in the supply of the cell lysate including themarker-tagged second proteins to the second substrate, the frequency ofthe fluorescent signal generation is just 8 times in 30 seconds.

However, as confirmed in FIG. 1, the cell lysate of the experimentalgroup-cell including the first proteins (HRas) with 41.3 nMconcentration that exceeds a threshold concentration (5 nM) is suppliedto the second substrate to induce binding between the first proteins andthe first protein-binding molecules. From that point, when the frequencyof the fluorescent signal generation with a specific wavelengthgenerated by a marker tagged to the second proteins is measured by usingan optical apparatus generating a near-field in a plurality of ROIs asits average value in the supply of the cell lysate including themarker-tagged second proteins to the second substrate, the frequency ofthe fluorescent signal generation is measured to be 20 times in 30seconds.

That is, while increasing the concentration value of the cell lysate ofthe experimental group-cell that is a tumor cell including the firstproteins (HRas), the average value of the frequency of the fluorescentsignal in a plurality of ROIs is measured. As a result, as shown in FIG.14, it has been seen that it is constantly maintained at 5 nM that is athreshold concentration value, and then increased after exceeding thethreshold concentration value.

Based on the threshold concentration value as mentioned above, theanalyst may calculate the ratio of activated first proteins among thefirst proteins included in the cell lysate of the experimentalgroup-cell.

In addition, the analyst may quantitatively calculate the ratio ofactivated first protein concentration included in the cell of thecontrol group and the activated first protein concentration included inthe cell of the experimental group by comparing the thresholdconcentration values in the control group-cell and the experimentalgroup-cell by repeating the experiments as mentioned above in thecontrol group-cell.

That is, the analyst measures the threshold concentration value in thecontrol group-cell under the same condition while slowly increasing theconcentration of the cell lysate of the control group-cell including thefirst proteins (HRas) along with the experimental procedure as mentionedabove, or before and after the experimental procedure to theexperimental group-cell in order to achieve the threshold concentrationvalue in the experimental group-cell.

Meanwhile, for increasing the concentration of the cell lysate in thecontrol group-cell, it is preferable to increase in the same ratio asthe ratio of increasing the concentration of the cell lysate in theexperimental group-cell.

That is, referring to FIG. 12, the analyst induces binding between thefirst proteins and the first protein-binding molecules by supplying thecell lysate of the control group-cell including the first proteins(HRas) with 3.5 nM concentration to the second substrate. From thatpoint, when the frequency of the fluorescent signal generation having aspecific wavelength generated by the markers tagged to the secondproteins is measured by using an optical apparatus generating anear-field in a plurality of ROIs as its average value in the supply ofthe cell lysate including the marker-tagged second proteins to thesecond substrate, the frequency of the fluorescent signal generation isjust 6 times in 30 seconds.

Meanwhile, from that point, when increasing the concentration of thecell lysate of the control group-cell, the frequency of the fluorescentsignal generation measured in a plurality of ROIs as its average valueis constant at 6 times in 30 seconds.

However, as confirmed in FIG. 13, the cell lysate of the controlgroup-cell including the first proteins (HRas) with 49 nM concentrationis supplied to the second substrate to induce binding between the firstproteins and the first protein-binding molecules. From that point, whenthe frequency of the fluorescent signal generation with a specificwavelength generated by a marker tagged to the second proteins ismeasured by using an optical apparatus generating a near-field in aplurality of ROIs as its average value in the supply of the cell lysateincluding the marker-tagged second proteins to the second substrate, thefrequency of the fluorescent signal generation is measured to be 7 timesin 30 seconds, that is increased.

From that point, while increasing the concentration of the controlgroup-cell lysate, the average value of a frequency of the fluorescentsignal is measured. As a result, as confirmed in FIG. 14, it has beenseen that it is distinctly increased after exceeding the thresholdconcentration value that is a 50 nM concentration.

From the above experimental results, it has been seen that the analystcan confirm the fact that the activated first proteins included in theexperimental group-cell that is a tumor cell are 10 times larger thanthose of the activated first proteins included in the control cell thatis a normal cell based on the fact that the threshold concentrationvalue in the experimental group-cell is measured to be 5 nM and thethreshold concentration value in the control group-cell is measured tobe 50 nM.

According to the present invention, each cell state and activationlevels of first proteins can be compared and confirmed by comparingafter varying of a type of cells supplying the first proteins forobserving interactions between the first proteins and the secondproteins.

Moreover, according to the present invention, the protein-proteininteractions can be analyzed at a single molecular level in the actualintracellular environment.

Moreover, according to the present invention, specific activated proteinconcentrations can be quantitatively measured in the cell of anexperimental group and the cell of a control group.

While the invention has been shown and described with reference tocertain exemplary embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the spirit and scope of the invention asdefined by the appended claims.

The invention claimed is:
 1. A method of analyzing protein interactionsbetween first proteins and second proteins, comprising: (a) preparing atleast two substrates including first substrate and second substrate, inwhich first protein-binding molecules that are biomolecules to be boundto the first proteins are attached to each of the substrates; (b)inducing binding between the first proteins and the firstprotein-binding molecules on the first substrate and the secondsubstrate, respectively, by supplying the first proteins as a controlgroup to the first substrate and supplying the first proteins as anexperimental group to the second substrate; (c) supplying cell lysatesincluding the second proteins tagged with markers to the first substrateand the second substrate, respectively; and (d) comparing and analyzingthe interactions between the first proteins and the second proteins onthe first substrate and the second substrate by measuring a fluorescentsignal having a specific wavelength generated by each of the markerstagged to the second proteins bound to the control group-first proteinsand the experimental-group first proteins, respectively, using anoptical apparatus generating a near-field, wherein the fluorescentsignal is measured cumulatively for a predetermined integral timeperiod.
 2. The method of claim 1, wherein in the step (b), the controlgroup-first proteins are normal cells and the experimental group-firstproteins are tumor cells.
 3. The method of claim 1, wherein in the step(d), the fluorescent signal having the specific wavelength is measuredin real-time under the presence of the cell lysates supplied to thesubstrate.
 4. The method of claim 1, wherein the step (b) includessupplying cell lysates including the control-group first proteins to thefirst substrate, and supplying cell lysates including theexperimental-group first proteins to the second substrate.
 5. The methodof claim 1, further comprising supplying a buffer solution to thesubstrate before the step (c).
 6. The method of claim 1, furthercomprising (e) repeating the steps (b) to (d) while increasing theconcentrations of the control group-first proteins and the experimentalgroup-first proteins in the step (b), by a predetermined ratio.
 7. Themethod of claim 6, wherein the step (d) includes comparing and measuringa frequency of a fluorescent signal having a specific wavelengthgenerated by the markers tagged to the second proteins bound to thefirst proteins in any configured observation region on the substrate. 8.The method of claim 1, wherein the first proteins that interact with thesecond proteins in the step (d) are activated first proteins among thefirst proteins bound to the first protein-binding molecules.
 9. Themethod of claim 1, wherein the step (d) further includes determiningfrequencies of binding and unbinding between the first proteins and thesecond proteins.
 10. The method of claim 1, wherein the step (d) furtherincludes determining a duration of forming one binding between the firstproteins and the second proteins and a time required for forming afollowing binding between the first proteins and the second proteins.11. The method of claim 1, wherein the first proteins are h-Rasproteins, the second proteins are Ras-binding domain (RBD) proteins ofC-Raf, the biomolecules and the first protein-binding molecules areantibodies, DNA, RNA or liposome having a specific components to bebound to the first proteins, and the substrate is a quartz slide coatedwith polyethylene glycol.